Process of dehydrating biological materials



United States of America as represented by the Secretary of AgricultureNo Drawing. Application August 13, 1958 Serial N0. 754,904

6 Claims. (Cl. 34-'-5) (Granted under Title 35, US. Code (1952), sec.266) A non-exclusive, irrevocable, royalty-free license in the inventionherein described, throughout the world for all purposes of the UnitedStates Government, with the power to grant sublicenses for suchpurposes, is grantedto the Government of the United States of America.

This application is a continuation-in-part of our prior pendingapplication Serial No. 628,205, filed Dec. 13, 1956, now Patent No.2,853,797, issued Sept. 30, 1958.

This invention relates to and has among its objects the provision oftechniques for dehydrating biological materials, particularly microbialcultures, whereby to produce solid, dry products which are capable ofbeing stored for indefinite periods of time without loss of activity.One object of the invention is the provision of processes of the abovetype wherein the dehydration is accomplished without damage to theviability of the culture so that the dry product is a nondeteriorating,instantly-available source of a viable culture which will proliferatevigorously and immediately upon exposure to moist conditions andnutritive materials. A further object of the invention concerns theprovision of procedures wherein the dehydration yields products whichare in a porous, disperse form which can readilybe reduced to a powderof extremely fine particle size without damage to the viability of themicroorganism. Another object of the invention concerns theaccomplishing of such techniques of dehydration in a rapid and eificientmanner requiring only the use of simple and inexpensive equipment.Another object of the invention is the provision of processes ofdehydration which are particularly characterized by their versatilityand adaptability to various conditions of temperature and the likewithout loss ofefficiency.

A particular object of the invention concerns the provision ofimrovements over the basic dehydration process disclosed in our priorapplication. These improvements involve the provision of procedureswhereby frozen particles undergoing dehydration are prevented fromcohering to one another or to the moisture adsorbent employed in thedehydration. By preventing such coherence, uniform and rapid dehydrationis ensured and contamination of the product with particles of theadsorbent is minimized. Basically, the improved results are attained bycoating the frozen particles with very finely divided inert materials asexplained in more detail below in the section designated B. Coating ofthe Frozen Particles.

In the fields of industrial, agricultural, and experimentalmicrobiology, it is often desired to preserve microbial cultures forextended periods of time. Maintaining such cultures on agar slants orsimilar media is not satisfactory because of the necessity for carefultemperature control, repeated transfers to fresh media, danger ofcontamination and so forth. Generally, it is preferred to dehydrate thecultures so that they will be converted into dry products which are ofmore stable character and which will withstand storage at ordinary2,897,600 Fatented Aug. 451-959 2 temperatures for long periods of time.In preparing such dry products, it is of utmost importance that theviability of the culture bemaintained; the organism is killed inthe'dehydration, the process is a futility. Further, for best results,the dehydration should not impair the viability of the culture to anyappreciable degree so that the dry culture will retain" its full vigorand will proliferate rapidly when exposedto-rnoist and nutritiveconditions.

organisms is more diflicult-than the spore-formers because thenonsporulating organisms contain only vegetative tissue which isintrinsically not well adaptet to resisting unfavorable conditions.

Usually microbial cultures are dehydrated by conven tionalfreeze-drying. procedures. On' an industrialscale this procedureinvolves filling the culture into metal trays and subjecting the filledtrays to refrigeration to freeze the cultures into a solid mass. Thetrays are then slid onto the hollow shelves of a vacuum shelf drier.-The drier'is then closed and'high vacuum equivalent to anabsolutepressure of about 50 microns of Hg or lessis applied. As the dehydrationproceeds a heated medium is circulated through the hollow shelves tosupply the heat needed for" sublimation of moisture from the frozenculture. Thus throughout the process the culture is main tained in afrozen state but as sublimation ofmoisture takes place, heat must besupplied to the culture to balance the cooling effect of thesublimation. If heat were not supplied, the cultureiwould become so coldthat the obvious that if the layer is extremely thin the dehydrator willbe used at onlya small fractionof its' intendedcapacity.) Duringt-h'edehydration it is" necessary; as pointed out above, to apply heatto the culture tosupply the heat of sublimation. This is donefby'circulatingv heated water through the hollow shelves; As a result; thebottom of the culture layer which is nearer to the heated shelf issubjected tomore heat than the upper partof the culture layer; (In thisregard it is to be noted that the culture is maintained in a solid,frozen state so that transfer of heat by circulating currents as wouldoccur in heating a fluid cannot take place.) 7 Under these conditions,the bottom portion of the culturefll ayer is dehydrated more rapidlythan the material in the upper portion and eventually becomes overheatedas its moisture content decreases and its temperature is no longer" keptin check by the cooling e'ifect of sublimation. In' order to dehydratethe remaining part of the culture in the upper portion of the layer, besubjected to the excessive temperature to serve as a" heat transfermedium to'transfer heat to the upper mate'- rial until it too isdehydrated; Naturally, this non-uniformity of heating leads to areduction of the vitality of the culture in that the material which hadbeen subjected" i I I the 'over'heating. of this part of the cultureproduces heat" decomposition products such as ammonia, hydrogensulphide, methanol I acetic acid, and other products of pyrolysis whichContact, the viable portions of the culture 'causin'g' such disadvan;tageous effects as inactivation of enzymes changes of to overheating isinactivated. Further,

pH- of cell contents, lysis of cellwalls and the like;

Naturally, if

In general the p'reparation of variable dry cultures from non-spore'forming the lower material must Conventional freeze-drying in additionto the problem of non-uniform heating, has the disadvantage thatexpensive apparatus is required. Thus the dehydrator must be of heavyconstruction to withstand the effects of vacuum and the vacuumsystem'must be of large capacity to continuously handle the tremendousvolumes of water vapor produced at the attenuated pressure involved.

It has been proposed by Tival (US. Patent 1,979,124) that biologicalinaterials such as meat, glandular material, milk, etc. be dehydrated bya process which involves freezing the material and subjecting it to veryhigh pressures while in such frozen condition to cause expression ofwater therefrom. The partially dehydrated materialis then admixed with asolid adsorbent such as silica gel and the resulting mixture maintainedunder a vacuum at subfreezing temperatures to cause transfer of moisturefrom the material to the adsorbent. This process has some advantagesover conventional freeze-drying since the vacuum apparatus isconsiderably simplified in that it need only pump fixed gasses such asair whereas the water vapor is adsorbed by the silica gel and is nothandled by the vacuum pump. However the procedure is not adapted topreserve the viability of microbial cultures. Thus an essential elementof the Tival process is that the frozen material be subjected to veryhigh pressures-about 2000 atmospheres (29,392 lbs. per sq. in) to pressout water. The subjection of, cellular material to such extreme pressurecauses its destruction by mechanical forces and by localized thermaleffects within the compressed mass. Thus the very fact that such highpressures are used will cause rupture of cell walls and expulsion ofprotoplasmic material from the cells. In addition, even if the press iscooled, heat transfer through the solid mass will be slow andineffective and uncontrolled so that localized heating efiects will takeplace within the mass due to friction between mechanical parts of thepress and especially due to internal friction between the particlesbeing compressed.

Another disadvantage of the Tival process is that it is not adapted tothe production of a porous, disperse product but rather one that is of adense, compressed nature. Thus in the Tival process an essential step issubiectingthe frozen material to extreme pressures. This compressionhaturally densities. the material by expulsion of water. Since theproduct of Tival is of a dense nature his not suited for variouspurposes. For example, a dense, productis not suitable for, theproduction of final products of an extremely fine particle size. Thusfor certain agricultural purposes it is necessary to prepare dehydratedmicrobial cultures in the form of a powder having a particle size ofabout 1 to 5 microns so that the particles can be dispersed in the formof clouds or aerosols having very low settling rates. In order toproduce active (viable) dry cultures of such fine particle size it isimperative that the dry product just prior to grinding be in an expandedor dispersed condition,that is, each particle of material must consistof a porous mass in which the solid components are spread out over aconsiderably larger volume than they would normally occupy if placednext to one another. When such expanded particles are sub ected togrinding, they can readily be transformed into particles of extremelysmall 'size, that is, on the order of l to 5 microns; In effect, thegrinding operation doesnot result in a division of the individual solidcom ponents which make up the particle but causes a separatron of thesesolid components from one another. Thus where each particle is composedof a group of bacterial cells, the individual cells being spread outovera volume eonsrderably greater than the volume they would occupy 1ftightly packed together into a dense mass, the grinding operation willhave-the effect of simply separating one cell from another, f rming aproduct of high viability sau t e s tleif any, division of individual(36115. l the amount of energy which is required to achieve thisseparation of individual elements is Qf low order -4 1 of magnitude sothat the material is not subjected to ex-- cessive mechanical stressesorthermal effects. This,'in turn, means that the viability of theproduct is retained and the size reduction is obtained easily andefliciently. On the other hand, if the particles presented to thegrinding operation are of a dense nature they will be difiicult to grindand the grinding will result in a division of individual components ofeach particle. Thus, where the material presented to the grindingoperation is in theform of particles consisting of closely packedbacterial cells, the grindin 'operation will result in division ofindividual cells with the result that the ground product will have a lowviability. Also with such dense particles, the amount of energy requiredto accomplish the size reduction is of a high order of magnitude so thatthe prod-- net is exposed to excessive mechanical stresses and thermaleffects. This causes loss in viability and makes the size reductionoperation more difiicult to accomplish.

The effect of the density of the product on the grind ing operation canbe further illustrated by the following description using stones inanalogy to bacterial cells:

' pression step ofTival means that the losses in viability A. Porouspr0duct.-Stones are each coated with a thin layer of cement thendeposited in a mold without applying any compressive forces, shaking, orother attempt to pack the material in the mold. The cement is allowed toharden then the product is removed from the mold. This product is foundto be a mass of stones cemented together but with many gaps andinterstices in it. Whenthe mass is struck with a hammer, it is observedthat the individual stones fiy apart and there is little breakage ofindividual stones.

B. Dense pmduct.St0nes are each coated with a thin layer of cement thendeposited in a mold applying compression and vibration to ensure packingtogether of the stones in the mold. The cement is allowed to harden thenthe product is removed from the mold. This product is found to be a massof stones cemented together in closely-packed relationship. When themass is struck with a hammer, nothing happens unless considerably moreforce is used than with product A. If such considerable force isapplied, the mass breaks and many of cleavage lines run throughindividual stones, that is, many of the stones are divided into pieces.

Another point to be made is that the dense products of Tival exhibitpoor rehydration characteristics in that when they are contacted withwater, the products will absorb the water at a slow rate. On the otherhand, a porous product is especially desirable where rapid rehydrationis a consideration. Porous products are admirably suited for rapidrehydration because when contacted with water the water is rapidly drawninto the crevices and interstices of the porous article.

As disclosed in our prior application, dehydration by sublimation can becarried out more efliciently and effectively by a procedure whichinvolves directly subjecting the material to be dehydrated in' a frozenstate to contact with a solid adsorbent material such as silica gel. Inour process the pressure treatment of Tival is elimimated with theresult that the dehydrated products areobtained in a porous, dispersestate and are thus in a condition admirably suited for further reductionin particle size without loss of viability and exhibit extremely highrates of rehydration. Also elimination of thecom advantages of theinstant process over that of Tival will be evident from the followingdescription.

The production of dehydrated products in accordance with'this inventionmay involve various alternative modi- I fications. In general,the-production includes a sequence of operations as outlined below:

(A) Initially there are prepared frozen particles of the material to bedehydrated. For'example, microbial cells aredispersed-inwaterand thisdispersion is formed into frozen particles. In the alternative,wholemicrobial cultures or other moisture-containing biologicalmaterials, whetherin'solid or liquid state, may be form'e'd' into frozenparticles.

'(B) The frozen particles are then coated with a'very finely dividedinert material.

(C) The-coated frozen particles are dehydrated by contacting them with asolid adsorbent under conditions of vacuum and temperature-control,usually with refrigeration for 'at least the first stage of thedehydration. Under these conditions,theparticles are dehydrated whileretaining their original dimensions thus to produce a dried producthaving an-extremely porous structure.

(D) Thedehydrated particles'and the solid adsorbent are then separatedfrom one another by sieving or the like.

'(E) The dehydrated particles are then subjected to grinding ifaiproduct of fine particle :size is desired.

'The :steps briefly described above are explained in more detail in thefollowing sections:

:A. 'PREPAMTION OF THE MATERIAL FOR DRYING :In the application of thisprocess to the dehydration of a microbialculture, for example, such aculture 'is first produced by conventional fermentation techniques suchas culturing the organism in question on a suitable liquid nutrientmedium under agitated submerged conditions'and un'der aerated ornon-aerated circumstances as required by the organism. Thecellularmaterial may be dehydrated separately or together with the residualnutrient medium. Preferably the cellular material is harvested-fromtheculture'asby the use of centrifugation or the like. The centrifuge cake(the bacterial cells) is then dispersed'in water usingefiicient'agitation, homogenization orthe'like to get a uniformdispersion. This dispersion is then formedinto frozenparticles by anysuitable procedure. For example, the dispersionis coated on a-r otatingdrum having a surface maintained below the freezing temperature. Theresulting frozen layer of the dispersion is then-r'emovedfrom the drumwith a scraper blade in the form of flakes. A preferred method forproducing the frozen particles involves introducing the dispersion inthe form of drops onto the surface of a cold liquid medium such asmethylene chloride, Ifluorinate'd hydrocarbon derivative,petroleumdistillate, or other inert volatile organic solvent of lowfreezingpoint. In this way the dispersion is frozen solid while it isstill in droplet form and the product is collected as a mass of pellets.This technique has the advantage that the size -of the pellets may bereadily controlled and the pellets'have uniform sizeand shape. Othermethods and equipment forproducing frozen particles from a liquidpreparation are well known in the art and can, of course, be used'inplace of those described.

The 'reason for dispersing the cellular material in water is to obtainthe dehydrated product in a dispersed or attenuated form. Thus if adispersion containing 10% solids and 90% water is frozen and thendehydrated under conditions that cause no shrinkage of the frozenparticles, the finalproduct will be of a very attenuated character, eachparticle being 90% air and 10% "cellular material on'a volumetric basis.The proportion of solids in the dispersioncan be varied in accordancewith the bulkiness desired in'the final dehydrated product, thegreaterthe degree of dispersiomthe'mor'e attenuated (less dense) will bethe dehydrated product. In manycases, the cellular material is dispersedin sufficient water to form a dispersion containing on the order ofabout 5 to about 20% solids.

The size of the frozen particles has an effect on de hydration.procedure, particularly on the rate of dehydrationand the rate at whichheat is generated '-dur ing the dehydration. 'Iihus smaller particleshave a greater ratio of surface :to total volume so that the'dehydration irate will be higher and the rate of heat generation(heatuof wetting of the adsorbent minus the heat of sublimation) will behigher. Conversely, larger particles will result in a lower rate ofdehydration and a lower .rate of heat generation. In general, to obtaina rapid dehydration effect without causing excessive generation of heat,the size of the piecesis kept in the range from about 5 to about A inch.

In producing the dispersion, the bacterial cells are usually dispersedin pure water. If desired, however, the water may contain addedingredients. Where the added ingredients are nonvolatile they will-ofcourse be present in the final product; if the added ingredients arevolatile they will be dissipated in the processing operation to a.greater or .lesser degree depending on their degree of volatility. Theadded ingredients may be, for example, dyes, :pigments, odorants,preservative agents, or other materials which will not adversely affectthe viability of the organism in question. In general such additives areused in small proportion, not to exceed about 20% "of the weight of thebacterialcells.

In the production of dehydrated viable micro-organisms, fortifyingsaltsmay be added to the dispersion to assist in preserving viability asdisclosed by Naylor and Smith, J. Bact., vol. 52', p. 565 (-1946). Forexample, in the case of Serratia marcescens, to the aqueous celldispersion onemay add about /2% each of ammonium chloride, thiourea, andascorbic acid.

B. COATING OF THE FROZEN PARTICLES The particles of frozen moistmaterial, however formed, are then-coated withavery finely divided,inert, water'- insoluble, solid material. An important consideration .ofthe "coating material is that it be in a very finely divided condition,by which is .meant that :theindividual units of the material 'be-onemicron-or less in-size, preferably less than =on'e-tenth micron.Materials in such physical condition will cohere properly to the frozenparticles even when subjected to abrasive forces so as to preventcoherence between individual frozen particles. Moreover, coatingmaterials of such physical dimensions areeffective to prevent coherencebetweenfrozen particles even when employed in small proportion, thatis,on the order ofi0.0l to 0.1% ofthe weight of the frozen particles. It

is naturallydesirable to limit the amount of coating material as much aspossible without losing its effectiveness thus to minimize the amount ofcoating material which eventually becomes mixed with thedehydratedproduct.

The-chemical composition of the coating material is of no consequence aslong -as the material has-the attributes of being inert to the-materialto be dehydrated, waterinsoluble, and solid. Preferably, the coatingmaterial should be :a substance which inaddition to the aforesaidcharacteristics .has-neithercthe ability to adsorb nor'absorb water.

A'preferre'd coating material is submicron silica. this :finely dividedstate, "the silica has no gel structure and has no significant watersorbing properties. 'Other coating material which may be used are, forexample, magnesium silicate, calcium silicate, calcium carbonate,aluminum oxide, aluminumisilicate, aluminum phosphate, carbon, ironoxides, sulphur, tungstemsilver, gold, etc.

It -'is obvious that in preparing 1 dried micro-organisms in accordancewith the invention, the coating material should be non-toxic'to'or-ganisms'if the dried product is intended to-be eventually used as astock for proliferanon toxic nature.

fin

The step of coating'the frozen particles prior to dehydration affordsmany advantages. In the absence of the coating, the frozen particlestend to adhere to one another during dehydration, Such aggregatedparticles lose moisture at a much slower rate than non-aggregatedparticles with the result that the final product exhibits a nonuniformmoisture content in that' the individual nonaggregatcd' particles havelower moisture content than the aggregated particles. Moreover,particles of the solid adsorbent'employed in the dehydration tend toadhere to uncoated particles of the frozen material. This results inhigh ash contents of the dehydrated products, that is, the dehydratedproduct is contaminated with adsorbent material. By employing thecoating procedure herein described both of these problems are obviatedinthat the frozen particles of material who dehydrated are kept separateso that uniform dehydration is achieved and coherence between thesefrozen particles and the particles of solid adsorbent is minimized sothat the dehydrated product contains a negligible proportion of materialderived from the adsorbent. A further advantage of the coating procedureof the invention is that if the frozen particles are inadvertentlysubjected to elevated temperatures whereby to cause surface thawing, theparticles will not cohere to one another.

C. DEHYDRATION OPERATION The coated, frozen particles of moist materialare then dehydrated by contacting them with particles of a solidadsorbent such as alumina, silica gel, calcined zeolite, and so forth;silica gel being preferred. The process is carried out under vacuum andat a temperature such that the particles of moist material remain in afrozen state. Usually a temperature well below'the freezing point ofwater is employed to prevent any liquefying from taking place. Thus thetemperature is usually kept below 20 F., preferably 10 F. or less. Ingeneral the dehydration may be accomplished as follows; The particles ofcoated, frozen moist material and the particles of solid adsorbent areplaced in a container. The container is evacuated and sealed and is thenagitated to thoroughly mix the contents. The container is then kept in aroom having a temperature below the freezing point of the particles ofmoist material and the container is continuously agitated or rolled tocause all the surfaces of the particles of moist material to come intocontact with the particles of adsorbent. The system is maintained undersuch conditions until the dehydration is completed. It is to be notedthat sealed to retain the vacuum. Thus the vacuum system need only pumpthe air out of the container; it is not used to remove water vapor fromthe system.

As noted above, the dehydration is carried out at a temperature lowenough to keep the material in a frozen condition. Usually it ispreferred to accomplish the temperature control by keeping the containerfor the frozen particles and adsorbent in a refrigerator during thedehydration operation. If desired, however, the container may beprovided with a jacket through which a refrigerated medium. iscirculated, or the container may be provided with internal coils throughwhich a refrigerant' is circulated.

In conducting the dehydration, the proportions of frozen material andsolid adsorbent are so regulated that there is suflicient of theadsorbentpresent to adsorb from the frozen material the amount ofmoisture needed 'to be abstracted to obtain the desired degree ofdehydramaterial in a first stage and completing the dehydration in asecond stage or. in several additionalstages.v Such technique isparticularly desirable wherethe material is ticles.

to be reduced to a very low moisture level, that is, about 1% moistureor less. In this stage-wise technique the frozen material and solidadsorbent are enclosed in a container which is then evacuated, sealed,and subjected to rolling while maintained in a refrigerated chamber aspreviously described. After the frozen materials are partiallydehydrated, the containeris opened and the partially dehydrated materialis separated from the partially hydrated adsorbent. The partiallydehydrated material and a fresh supply of adsorbent are placed in thecontainer which is then evacuated, sealed and subjected to rolling tointermix the contents while maintained at a suitable temperature. Thedehydration may be completed in two stages or one may apply three ormore stages. One advantage of this stage-wise technique is that itpermits dehydration to very low moisture levels, that is, to about 1%moisture or less. Such results cannot be obtained in a single-stagedehydration for this reason: As the dehydration proceeds, the adsorbenttakes up water and'develops an increasing vapor pressure which limitsthe extent of dehydration, that is, the dehydration process would ceasewhen the vapor pressure of the partially dehydrated material and thevapor pressure of the adsorbent reached the same levelthe system wouldthen remain in a state of equilibrium. However, when the partiallyhydrated absorbent is removed and replaced with fresh adsorbent thislimiting factor is eliminated and the complete dehydration can beachieved.

It has also been found in connection with the stagewise technique ofdehydration that in the later stages of dehydration, the cooling stepmay he often eliminated. Thus in many instances, after the material hasbeen dehydrated to the extent that its moisture content is about 10% orless, the volume of the particlesis set and their exposure totemperatures above the freezing .point will no longer cause shrinkage intheir volume. Thus if in the first stage of dehydration the particlesare reduced to a condition of about 10% moisture or less, the secondstage ofdehydration may be conducted at room temperature or even atslightly elevated temperatures, for example, 70 to F. with no shrinkageof volume. The advantage of using temperatures above freezing is thatthe final dehydration is obtained more rapidly yet withoutdamage to thephysical state of the material. In any event, the heat applied duringthe last stage of dehydration should be not so high as to damage theviability of the organism being dehydrated. In most cases, thetemperature range cited will not adversely aifect the viability at thelow moisture levels existing in this stage of dehydration. It is obviousthat conducting the dehydration under room temperature conditions orwith moderate heating is only possible after the particles have beendehydrated to the extent that their volume and shape are set. Thus, ifit takes several stages of dehydration to bring the material to thisdimensionally stabilized condition than the usual cooling will be neededin all these stages but room temperature or moderate heating can beapplied in the following stage or stages.

By proceeding as above described the particles of frozen moist materialare dehydrated without substantial loss of viability of the organism andwithout shrinkage of the particles. Thus during the dehydration theparticles retain their original volume, the space in each particleinitially occupied by ice crystals being replaced by voids. Thedehydrated particles are thus of an extremely porous, attenuated,friable structure and have a density which is but a small fraction ofthe density of the original frozen particles. Depending on the solidscontent of the original dispersion, the dehydrated particles will havedensity of about one-fifth to one-twentieth of the density of theoriginal frozen par- Because the dehydrated particles have such a porousstructure they are eminently suitable for being reduced to a finepowder, that is, one in which the individual grains have about the samesize as the individual bacterial cells, e.g., about 1 to 5 microns indiameter. In reducing the porous dehydrated particles into a powder, theamount of energy necessary to achieve thesize reduction is of'a loworder of magnitude, as compared .with reducingthe size of denseparticles. As a result, the size reduction is'accomplished easily andwith no substantial loss of viability.

This invention includes within its scopeseveral novel modificationswhich may be applied in connection with the basic technique ofadsorption sublimation dehydration described above. These alternativefeatures are advantageous as minimizing localized heating of the frozenmaterial or as minimizing the deleterious'effects of such localizedheating. These modifications are particularly useful in the dehydrationof frozen particles which have a diameter of less than about 34 inch, inwhich case the rate of heat generation during dehydration is especiallyhigh. One of the novel modifications involves a plan of procedurewherein the frozen particles and the particles of'adsorbent are keptunmixed, or mixed as little as pos sible, prior to application ofvacuum. A specific mode of applying this technique involves thefollowing steps: The frozen particles and the particles of adsorbent areplaced in separate piles in the vacuum tank, the tank is evacuated,sealed under vacuum, and the materials in the tank are then mixed. Thedehydration is then carried out as previously described. The advantagesof such procedure are explmed as follows: Where the frozen particles andparticles of adsorbent are mixed under atmospheric pressure conditions,the: heat liberated due to rapid adsorption of moisture by the adsorbentmay cause surface melting of the particles which will result .in'a'shrinkage of the size of the particles and a final result will'be thatthe dehydrated particles will have increased density. On the other hand,when proceeding in accordanceiwith this modification of the inventionthe frozen:

particles because liquid water does not exist at the low pressureprevailing; Water can exist. only as ice or water vapor. Further,because of this physical phenomenon, the heat liberated as heat ofadsorption when moisture is adsorbed by the silica gel is dissipatedatleastin part to supply heat needed. to cause sublimation. of moisturefrom the material being dehydrated, this utilization of heat furtherpreventing undue temperature rises. Although it is preferred to keep thefrozen particles and silica :gel separate until vacuum is applied, it isoften diffieult to actually do this because of space limitations. in.the vacuum tank, etc. In such .case premature mixing: may blepreventedin whole or in. part by layering the material in the tank, that is, bydepositing. one material on, top of the other inseparate layers or byinserting a temporary divider between the layers which divider isturned. to one side out of the way after vacuum is applied to. thetank.. In any case, whether layering, mechanical separating, or the likeis used, it is advisable to close thetank and apply the vacuum asrapidly as possible. After a suitable vacuum has been drawn on the tankthe vacuum pump may be operated continuously. However it is generallypreferred to seal the tank after the proper level of vacuumhas beenreached and disconnect the limation: dehydrationoperation involvesinitially using.

a, silica gel. adsorbent which has been partially hydrated, that is, asilica gel which containsv about.

1.0% moisture. This has. the benefit that less. heat is generated due toadsorption of moisture by the gel where.-v

of solid. adsorbent.

sieved the particles of adsorbent will remain on the sieve. whereasthedehydrated particles will pass through the- In the alternative, thematerial may be prepared by the danger of reducing the viability of theproduct or causing it to shrink in volume is lessened. In applying this.mode of operation, the frozen particles of bacterial cell dispersionare placed in a container-together with particles of silica gel, thelatter having a moisture content of about 10%. The tank isevacuated andsealed and agitated while kept under refrigeration to preservetheuparticles in the frozen state. The benefit of using a partiallyhydrated silica gel is explained further as follows:

The amount of heat released when silica gel is mixedwith ice varies withthe amount of moisture on the silica gel. Thus the amount of heatreleased when silica gel operates in the range of 0 to 5 percentmoisture is greater than when an equal increment of moisture is addedbe-' tween 5 and 10 percent or 10 and 15 percent moisture. Starting adrying operation with silica gel containing 10% moisture thus providesmilder drying conditions (less.

generation of heat) than when fully dehydrated silica gel is employed.

Another expedient which may be employed to prevent undue temperaturerise during dehydration involves astepwise addition of the adsorbent.Thus the dehydration may be carried out in the following manner: Thefrozen particles and an amount of adsorbent less thanv would berequiredto dehydrate the particles are enclosed dration may involve twoadditions of adsorbent or as;

many additions as desired. The advantage of this technique is that thetotal. heat given off by the adsorption of moisture on the silica: gel.is spread out over a greater period. of time than where the neededamount of silica gel is applied in one batch. Since the heat is givenoff at a decreased rate, the danger of local overheating isminimized.Where the dehydration is conducted in a:

stage-wise manner, the stepwise addition of adsorbent is preferablyemployed during the initial stage (or stages):

of dehydration necessary to bring the particles to such a moisture levelthat their dimensions become fixed.

D. SEPARATION OF DEHYDRATED PRODUCT AND ADSORBENT After the dehydrationis completed, the dehydrated particles and particlesof adsorbent areseparated from one another. sieving operation. and adsorbent are shakenon the sieve, the pieces of dried material because of their friablenature break up enough to pass throughthe sieve whereas the particles ofad-- sorbent will remain on the sieve. Another plan which may be used isexplained as follows: In conducting the,

dehydration, the dimensions of the frozen particles are selected to besmaller than the dimensions of the particles When the. dehydratedproduct is sieve. in the form of frozen particles which are larger thanthe particles of adsorbent in which case the dehydrated particles willbe retained on the sieve and the adsorbent'will Another; technique is touseas the adsorbent, silica gelor the like: containing asmall'proportion of a material- This can be acomplished, for example, bya.

In some cases when. the dried material- I aration of the V 1 1 havingmagnetic properties such as magnetite. The composite, final product canthen be separated into its components by the application of conventionalmagnetic separating devices.

E. DISINTEGRATION OF DEHYDRATED PRODUCTS The dehydrated products, ifdesired in fine-particle form, may be subjected to attrition employinghigh speed grindersor other known devices suitable for producing finepowders. As noted above, thedehydrated products of this invention are ina friable, porous, attenuated form so that they are capable of beingreduced to fine. particles with the application of moderate degrees ofenergy and without substantial destruction of viability because theproduction of the fine particles involves essentially sepindividualcells and not cleavage of individual cells.

V F. GENERAL CONSIDERATIONS As noted above, this invention isparticularly concerned with the production of dehydrated solid productsfrom microbial preparations. The products are non-deteriorating,instantly available sources of the organism in a viable state so thatthe organism will proliferate vigorously immediately upon contact of theproduct with moist and nutritive conditions. Also the invention enablesthe production of such products in the form of extremely fineparticles,'having a particle size of about 1 to 5 microns, that is,essentially the same size as the cells of the organism. Such finelydivided product is of particular usefulness in agriculture as theseparticles may be dispersed in the form of clouds or aerosols having verylow settling rates. The application of microbial preparations in aerosolform is useful for combatting insect pests on plants and in soils andfor control of plant pathogens on plants and in soils. In suchapplications, the microbial organism which is capable of attacking theinsect or disease-producing organism in question is dehydrated andreduced to fine particles in accordance with this invention. Theparticles are then applied by known devices as an aerosol to theinfected plants or soil. Examples of microbial organisms and the pestsor diseases against which they are effective are as follows: Bacilluspopilliae for infecting Japanese beetles with type A milky disease;Aerobacter aerogenes var. acridiorum for eradication of locusts(Schistocerca) the fungus Be auveria bassz'ana for control of theEuropean corn borer and the codling moth; various species of the fungiEmpusa and Entomophthora for the control of aphids, leaf-hoppers, flies,grasshoppers, and so forth; cultures of the streptomycin-producingorganism Actinomyces griseus for the control of halo blight (Pseudomonasmedicaginis var. phaseolicola) and common blight (Xantlzomonas phaseoli)on beans. It is obvious that other bacterial, fungal, antibioticpreparations and so forth may be reduced to dry, fine particle form inaccordance with this application. The process of the invention is alsouseful for drying bacterial cells for use in biological assays.

Although this invention is particularly adapted to the dehydration ofmicrobial cultures, it can be applied to any kind of biologicalmaterial. Thus one may utilize the invention for the dehydration offoods such as fruits, vegetables, meat, fish, eggs, milk, soups, fruitjuices, and so forth. Also the process of the invention may be utilizedfor the dehydration of protein solutions, therap'eutic biologicalpreparations, antibiotics, glandular preparations, sera, enzymesQyeast,vitamin concentrates, etc. In all such cases, the use of the dehydrationprocess of this invention has the advantage that the removal of moistureis accomplished without damage to the essential components andcharacteristics oftheoriginal material. For example, in'the case offoods, such essential attributes as flavor and vitamin content areretained; in'

. v 12 the case of proteins, enzymes, therapeutic products, antibiotics,vitamin concentrates, etc., such essential attributes as chemicalidentity, enzyme activity, nutritive value, etc. are retained. In short,the dehydration is attained without deterioration of the labileconstituents of the original material. In applying the dehydration tosolid materials such as meat, fruits, vegetables, etc. the material maybe cut up into small particles, then frozen, coated and dehydrated asdescribed above. If it is desired to make final product of a more porousand disperse character then the solid material may be comminuted to forma pulp or slurry which is then dispersed in Water, the dispersion beingformed into frozen particles, coated, and dehydrated as described.Obviously by increasing the degree of dispersing (using more water perpart of startingmaterial) the product will be of a lighter and moredisperse character. Where the starting material is a liquid such as ananimal 'or plant serum the'liquid may be frozen directly and formed.into particles for application to the dehydra- 'tionoperation or it maybe admixedwith water before freezing to get a final product of lessdense nature. The invention is further demonstrated by the followingexamples. 7

- Example I I A culture of Serratia marcescens was prepared, thebacterial cells being then separated by centrifugation and washed withwater. The cells were then agitated with water to produce a dispersionof the bacterial cells, the dispersion containing 10% solids.

The dispersion was caused to fall while in droplet form into a bathcontaining a mixture of trichloro-monofiuoromethane andtrichlorotrifluoroethane, maintained at minus 50 C. The pellets offrozen aqueous bacterial dispersion, having a diameter of one-eighthinch, were separated from the bath and allowed to stand at minus 18 C.to permit vaporization of fluorinated hydrocarbons from the pellets.

Four -gram batches of the frozen pellets at 0 F. were placed inindividual screw-cap containers. Two of the batches were treated with0.05% by weight of submicron silica (not in gel form). The treatmentinvolved adding 0.05 gram portions of the submicron silica to each oftwo of the containers and shaking for a few seconds. The remaining twosamples of frozen pellets were not coated thus to provide a control.Each of the'four batches of frozen pellets were dehydrated as followsiOne hundred grams of the pellets and 400 grams of 10 to 14 mesh silicagel (at 0 F.) were placed in a cylindrical metal container equipped witha sealable lid carryinga conduit and valve arrangement. The containerwasclosed and exhausted to 1 mm. Hg through the conduit. The source ofvacuumwas then disconnected and the valve closed to thus seal thecontainer. After shaking of the container to thoroughly mix thecontents, it was placed on a roller device 'whereby the container wasrotatedabout its axis at a speed of about 0.5 r.p.rn. This mixing byslow rotation was continued for 4 hours at 0 F.

At the end of this time the four containers were opened and thedehydrated products separated from silica gel by screening. It wasobserved that the pellets of bacterial dispersion had retained theiroriginal spherical shape. However, the product which had not been coatedshowed some clusters formed by coherence of individual pellets duringdehydration. In the product 'wherein the pellets had been coated therewere no clusters present. Observation of the products under a low-powermicroscope disclosed smallrpieces of silica gel stuck to the pellets andthe clustered pellets of the uncoated product: The coated pelletsexhibited little if any of the silica gel on the pellets. t I "Ashingprocedures conducted on the dried pellets gave there results' M 13Product: Silica content, percent Coated 0.15 Uncoated 0.45

Example 11 A culture of Leucanostoc mesenteroides was prepared andcentrifuged to separate the cellular material. This material wasagitated in sufficient Water to form a dispersion of 11% solids and thedispersion was neutralized to pH 7.0 with 1% KOl-I solution.

The bacterial dispersion was formed into frozen pellets in the samemanner as described in Example 1.

Two lOO-gram samples of the frozen pellets at F. were weighed intoseparate screw-cap bottles. One batch of pellets was coated by shakingwith 0.05% of submicron silica (not in gel state). The other batch ofpellets was left uncoated to provide a control.

The coated and uncoated frozen pellets were dehydrated exactly asdescribed in Example 1.

After drying and separating the silica gel and pellets, the presence ofclumps was observed in the uncoated product. Screen separations showedthat 12% of the pellets present in the uncoated sample were clustered inclumps of 3 or more pellets. No clumping was observed with the coatedsample.

Example III A culture of Serratia marcescens was prepared, the bacterialcells being then separated by centrifugation and washed with water. Thecells were then agitated with water to produce a dispersion of thebacterial cells, the dispersion containing 19% solids.

The dispersion was caused to fall while in droplet form into a bathcontaining a mixture of trichloro-monofluoromethane andtrichlorotrifluoroethane, maintained at minus 50 C. The pellets offrozen aqueous bacterial dispersion, having a diameter of one-eighthinch, were separated from the bath and allowed to stand at minus 18 C.to permit vaporization of fluorinated hydrocarbon from the pellets.

The frozen pellets were coated by shaking them for a few seconds with0.05% of their weight of submicron silica (not in gel state).

One hundred grams of the coated pellets and 400 grams of 6 to 12 meshsilica gel were placed in a cylindrical metal container equipped with ascalable lid carry-- ing a conduit and valve arrangement. The containerwas closed and exhausted to 1 mm. Hg through the conduit. The source ofvacuum was then disconnected and the valve closed to thus seal thecontainer. After shaking of the container to thoroughly mix thecontents, it was placed on a roller device whereby the container wasrotated about its axis at a speed of about 0.5 r.p.m. This mixing byslow rotation was continued for 4 hours at 0 F.

At the end of this time the container was opened. It was observed thatthe pellets of bacterial dispersion retained their original size. Bythis operation the density of the pellets was reduced from 1 gram percc. to about 0.1 gram per cc. There was no evidence of clumping ofindividual pellets. The contents of the container was placed on a35-mesh sieve and subjected to shaking to break up the pellets. Thepellet material passed through the sieve leaving the silica gelparticles on the sieve. The pellet material, that is, the bacterialpreparation, had a moisture content of 10%.

The material of 10% moisture content from the preceding step wassubjected to a second drying in the same general manner as in the firststage. Thus the material was placed in the container with 5 times itsweight of silica gel. The container was evacuated and subjected to aslow rolling action for 3 hours at 70 F. The container was then openedand the product screened through a -mesh sieve. In this case the sievingoperation was conducted in a chamber containing air at low humidity toprevent uptake of moisture by the dried material. The dried bacterialpowder passing through the sieve had a moisture content of 1% and aviability of 68%. A sample of this product ground in a high-speedgrinder yielded a product with a mass mean diameter of 4.2 microns asdetermined by sedimentation.

Having thus described our invention, we claim:

1. In a process for dehydrating an organic material wherein frozenparticles of water-containing organic material are contacted with asolid adsorbent while maintained under vacuum and at a temperatureregulated to keep the particles in a solid state during the dehydration,the improvement which comprises coating the particles with a veryfinely-divided, inert, water-insoluble, solid material prior tocontacting the particles with the solid adsorbent whereby to inhibitcoherence of the particles with one another and with the solidadsorbent.

2. The process of claim 1 wherein the solid material is silica in asubmicron state of subdivision.

3. The process of claim 1 wherein the organic material is microbialcells and the solid material is silica in a submicron state ofsubdivsion.

4. A process for dehydrating a biological material which comprises:forming a water-containing biological material into frozen particles;coating the frozen particles with a very finely-divided, inert,water-insoluble, solid material; subjecting the coated, frozen particleshaving their original moisture content and original volume todehydration by contacting them .with a solid adsorbent under vacuum andat a temperature at which the particles remain in a solid state, thetemperature being at a subfreezing level for at least the first part ofthe dehydration; and separating the dehydrated particles from theadsorbent.

5. The process of claim 4 wherein the solid material is silica in asubmicron state of subdivision.

6. The process of claim 4 wherein the biological material is microbialcells and the solid material is silica in a submicron state ofsubdivision.

References Cited in the file of this patent UNITED STATES PATENTS2,446,075 Blair July 27, 1948 2,723,954 Young Nov. 15, 1955 2,853,797Graham et al. Sept. 30, 1958

1. IN A PROCESS FOR DEHYDRATING AN ORGANIC MATERIAL WHEREIN FROZENPARTICLES OF WATER-CONTAINING ORGANIC MATERIAL ARE CONTACTED WITH ASOLID ADSORBENT WHILE MAINTAINED UNDER VACUUM AND AT A TEMPERATUREREGULATED TO DEEP THE PARTICLES IN A SOLID STATE DURING THE DEHYDRATION,THE IMPROVEMENT WHICH COMPRISES COATING THE PARTICLES WITH A VERYFINELY-DIVIDED, INERT, WATER-INSOLUBLE, SOLID MATERIAL PRIOR TOCONTACTING THE PRATICLES WITH THE SOLID ADSORBENT WHEREBY TO INHIBITCOHERENCE OF THE PARTICLES WITH ONE ANOTHER AND WITH THE SOLIDADSORBENT.